Dextran and Food Application

Abstract

An increasing consumer trend towards healthy and additive-free food has made dextran from food grade lactic acid bacteria (LAB) an attractive solution. Dextrans are homopolysaccharides of d-glucose produced by extracellular dextransucrase released from LAB of the genera, viz., Leuconostoc, Lactobacillus, Streptococcus, Weissella, and Pediococcus. Dextrans have been known for their viscosifying, emulsifying, texturizing, stabilizing attributes in food applications. Dextran has the potential to be recruited as a novel ingredient replacing the commercial hydrocolloids in bakery and other food industries. Prebiotic oligosaccharide production by hydrolysis of dextran is a rather new field, garnering research and industrial attention. The applications, available sources, preparation, and characterization of dextran and problems associated with its use have been discussed. This chapter also highlights the key developments in recent times and discusses the importance of bio-prospecting novel dextran-producing isolates from biodiversity.

Keywords

1 Introduction

The progressively increasing demand of natural polymers for various industrial applications has led to the exploration of microbial exopolysaccharide (EPS) in recent years. Among several EPS, dextran has gained worldwide recognition due to its biodegradability and biocompatibility properties (Patel et al. 2010; Aman et al. 2012; Varshosaz 2012). The EPS dextran was first discovered by Louis Pasteur (1861) as a microbial product in wine. Scheilber in 1874 confirmed that this microbial polysaccharide has a positive (dextrorotatory) optical rotation with the empirical formula (C6H10O6)n and therefore named as “dextran.” The physiological roles of EPS in the microbial host are not yet completely understood, but they are involved in protection against dehydration, pathogenicity, biofilm formation, and quorum sensing. The presence of a dextran layer around the bacterial cell may have paramount effects on the cellular diffusion properties. Dextran has gained importance owing to its applications in the food, pharmaceutical, biomaterial, photo film manufacturing, and fine chemical industries. A large number of lactic acid bacteria (LAB) are known to produce dextran.

Hehre in 1941 reported the first cell-free synthesis of dextran using sucrose as the substrate from enzyme dextransucrase. Dextransucrase (sucrose: 1,6-α-d-glucan 6-α-glucosyltransferase) is the key enzyme that catalyzes the synthesis of dextran from sucrose. Dextrans generally vary in their molecular weight, spatial arrangement, type and degree of branching, and length of branched chains, depending on the source of strains and also on the cultivation conditions. A survey of dextrans from 96 strains (primarily Leuconostoc mesenteroides) demonstrated that the amount of α-(1 → 6) linkages in a specific dextran can vary from 50 % to 97 % of the total glycosidic linkages (Jeanes et al. 1954). Apart from α-(1 → 6) linkages in main chain, dextrans also contain α-(1 → 2)-, α-(1 → 3)-, and α-(1 → 4)-branched linkages. Branched dextrans have also been reported to possess prebiotic (Das et al. 2014) and anticancer potentials (Shukla and Goyal 2013).

2 Sources of Dextran

Dextran occurs naturally in small amounts in foods, such as refined crystalline sugar, maple syrup, sauerkraut juice, and honey, and also as a component of dental plaque. Dextran is synthesized by the action of bacterial enzyme, dextransucrase, on sucrose. Dextransucrase is the sole industrial enzyme used in the commercial production of dextran and is produced by LAB of genera, viz., Leuconostoc, Streptococcus, Lactobacillus, Pediococcus, and Weissella. The structure of each type of dextran depends on the microbial strain and hence on the specific dextransucrase. To date, commercial dextran is produced from Leuconostoc mesenteroides NRRL B-512F and serves as a model in studying the structure of dextran and the mechanism of its biosynthesis by dextransucrase (Robyt et al. 2008; Siddiqui et al. 2014).

The amount of dextran produced however is practically insufficient to meet the dextran requirements of the various industries; hence, there is the need for the isolation and characterization of hyper dextran-producing LAB. Several examples of dextran with their linkage pattern from LAB isolated from various food sources are mentioned in Table 1.

Table 1

Dextrans with their linkage pattern from different LAB isolated from various food sources

3 Dextransucrase

Dextransucrases (EC. 2.4.1.5) are the sole industrial enzymes used in the commercial production of dextran (Parlak et al. 2013). Dextransucrase is classified in the family of glucansucrase, and most of the enzymes classified in this family use sucrose as the d-glucopyranosyl donor to synthesize α-d-glucans of high molecular mass with the concomitant release of d-fructose. They are also referred to as glucosyltransferases (GTF) because they synthesize α-glucan polymers using the glucose unit of sucrose (Leemhuis et al. 2013). Glucansucrases have been listed within family 70 glycoside hydrolase (GH70) in carbohydrate-active enzyme database (http://www.cazy.org/Glycoside-Hydrolases.html) based on sequence similarity (Cantarel et al. 2009; Vujicic-Zagar et al. 2010). They are evolutionarily closely related to the enzymes such as amylosucrase, cyclodextrin glucanotransferase, amylomaltase, and α-amylase from families GH13 and GH77 (Cantarel et al. 2009). Together with the families GH13 and GH77 enzymes, they form the clan GH-H (Vujicic-Zagar et al. 2010; Leemhuis et al. 2013). However, glucansucrases are much larger enzymes (~1,600–1,800 amino acid residues) than GH13 and GH77 (~500–600 amino acids), and they contain an N-terminal domain of variable region of unknown function (Vujicic-Zagar et al. 2010). Glucansucrases usually display a (β/α)8 barrel-shaped protein folding pattern and the acid–base-assisted substrate catalysis via a double-displacement (retaining) mechanism. GH70 enzymes are transglucosylases produced by LAB of genera, viz., Streptococcus, Leuconostoc, Weissella, and Lactobacillus (Monchois et al. 1999; Ito et al. 2011; Leemhuis et al. 2013). Four distinct types of GH70 glucansucrases have been identified based on the polysaccharides produced by them (Andre et al. 2010).

The enzyme dextransucrase synthesizes dextran from sucrose with concomitant release of fructose by double-displacement mechanism (Fig. 2). In the first stage of double-displacement reaction, α-(1 → 2) glycosidic linkage of sucrose is cleaved with the release of fructose, and a glucosylenzyme intermediate is formed, in which the glucosyl unit is covalently attached to the catalytic nucleophile via a β-glycosidic linkage. In the second stage of reaction, the covalently bound glucosyl moiety is transferred to the accepting nonreducing end of sugar of a growing glucan chain, with reformation of the α-glycosidic bond (Leemhuis et al. 2013).

4 Preparation of Dextran

Dextran is produced commercially by cultivating L. mesenteroides strains in situ in growth medium supplemented with sucrose and in vitro by using purified dextransucrase with sucrose as a substrate (Leemhuis et al. 2013). The dextran of desired molecular weight can be achieved by the direct enzymatic synthesis using purified dextransucrase, which allows more control over the reaction conditions as compared with the fermentative synthesis (Falconer et al. 2011). The production of dextran by dextransucrase from LAB is affected by factors like temperature, aeration, and concentration and type of medium components (Tsuchiya et al. 1952; Lazic et al. 1993; Purama and Goyal 2005; Bejar et al. 2013). Dextran production is also influenced by solubility, viscosity, nitrogen, phosphorus, and ash content of the medium (Jeanes et al. 1954). The molecular weight of dextran is inversely proportional to the concentration of enzyme and directly proportional to the concentration of sucrose. Moreover, the molecular weight of dextran increases as the temperature increases from 20 °C to 30 °C (Falconer et al. 2011). Several physical and chemical techniques such as UV irradiation (Patel and Goyal 2010; Agrawal et al. 2011; Siddiqui et al. 2013), ethyl methanesulfonate (Kim and Robyt 1994) and N-methyl-N′-nitro-N-nitrosoguanidine (Kitaoka and Robyt 1998), and site-directed mutagenesis (Funane et al. 2005) have been used for the enhancement of dextransucrase and dextran production from various LAB.

A single dextransucrase can catalyze the synthesis of several types of dextran linkages, thereby permitting the formation of a branched polymer (Neely and Nott 1962; Smith et al. 1994). Certain bacterial strains have been shown to produce dextrans of different structures due to the elaboration of different dextransucrases (Cote and Robyt 1982; Zahnley and Smith 1995). Thus, the structure of each dextran is a characteristic of the specific dextransucrase produced by a specific microbial strain (Jeanes et al. 1954; Vettori et al. 2012; Kothari and Goyal 2013).

5 Properties of Dextran

5.1 Physicochemical Properties

Dextran polymers have a remarkable diversity in chain length and in physicochemical properties due to the variation in degree of branching in their glucose backbone. In general, dextran is readily soluble in water, dimethyl sulfoxide, formamide, ethylene glycol, and glycerol but insoluble in monohydric alcohols, e.g., methanol, ethanol, and isopropanol, and also most ketones, e.g., acetone and 2-propanone. However, the water solubility of dextrans depends upon the branched linkage pattern. Linear dextrans have high water solubility, and the aqueous solutions behave as Newtonian fluids. However, some branched dextrans showed shear rate thinning effect, exhibiting non-Newtonian pseudoplastic behavior (Das and Goyal 2014). Viscosity of dextran solution depends on its concentration, temperature, and molecular weight. As dextran is a neutral polysaccharide, the viscosity is not significantly influenced by changes in pH or salt concentration. Dextrans with >43 % branching through α-(1 → 3) linkages are water insoluble. Dextrans have molecular weight in the range of 3–500,000 kDa. Dextrans with a molecular weight of 2,000–10,000 kDa exhibit the properties of an expandable coil, and at lower molecular weights (<2,000 kDa), dextran is more rodlike. Low molecular weight dextrans (40, 60, and 70 kDa) are generally preferred in clinical applications (Naessens et al. 2005). High molecular weight dextrans with few branched linkages are required for the application in sourdough (Lacaze et al. 2007). The surface morphological studies of dextran revealed a porous structure (Shukla and Goyal 2013; Das and Goyal 2014). The dextran has excellent thermal stability with degradation temperature ~300 °C (Das et al. 2014; Rao et al. 2014).

6.1 Isolation of Pure Dextran

The structural analysis of dextran starts with its isolation in pure form in such a way that the chemical and physical properties are not affected (Leemhuis et al. 2013). The recovery or purification from culture medium or enzymatic reaction mixture generally involves the following steps: (i) cell removal by centrifugation or filtration in case of culture medium, (ii) dextran precipitation from the cell-free supernatant or enzymatic reaction mixture by the addition of water-miscible organic solvents (e.g., ethanol, acetone, etc.), (iii) re-precipitation and dialysis of dextran, and (iv) size-exclusion chromatography (SEC) of dextran (Vettori et al. 2012; Shukla et al. 2014; Das et al. 2014). The high molecular weight dextran can be purified by SEC; however, low molecular weight dextran can be purified by ultrafiltration.

6.2 Molecular Weight Analysis

The molecular weight (MW) of dextran can be determined by colorimetric determination of reducing sugar, viscometry, hydrodynamic chromatography (HDC), high performance size-exclusion chromatography coupled with refractive index detector (HPSEC-RI), or with multi-angle laser light scattering (HPSEC-MALLS) (Leemhuis et al. 2013). However, higher branched polysaccharides are not well fractionated by means of classical SEC due to their shear scission, low exclusion limit (Cave et al. 2009), and limited resolution (Vilaplana and Gilbert 2010). Avoiding these problems, asymmetrical flow field flow fractionation (AF4) has emerged as a powerful technique for determination of the macromolecular structure of high molar mass branched biopolymers up to 108 Da (Rolland-Sabate et al. 2011). Recently, AF4 coupled with MALLS have been used to determine the MW of hyperbranched α-glucans (2 × 106 to 4.3 × 107 Da) (Rolland-Sabate et al. 2014).

6.3 Structural Unit and Glycosidic Linkage Analyses

The monosaccharide composition of dextran can be determined by acid hydrolysis followed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) (Mopper et al. 1992; Kothari and Goyal 2013; Shukla et al. 2014). The structural characterization of dextran can also be accomplished by well-known techniques such as one-dimensional (1H and 13C) or two-dimensional (TOCSY, NOESY, ROESY, and HMQC) nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM) (Maina et al. 2008; Bounaix et al. 2010; Vettori et al. 2012; Shukla et al. 2014). To elucidate the structure of larger dextrans, it is often necessary to prepare oligosaccharides from the native polysaccharide by mild acid or enzymatic hydrolysis (Leemhuis et al. 2013). These oligosaccharides are then identified by thin layer chromatography (TLC), high performance liquid chromatography with refractive index detector (HPLC–RI), HPAEC–PAD, mass spectrometry coupled with liquid or gas chromatography (LC–MS or GC–MS), and FTIR and NMR spectroscopy (Naessens et al. 2005). Finally, by combining the dextran analysis data as well as structural information of the oligosaccharides produced will unveil all the structural features of the dextran (van Leeuwen et al. 2008).

7 Food Applications of Dextran

Dextran has been studied as a food ingredient since the 1950s. The US Food and Drug Administration (US FDA) currently lists dextran as GRAS (generally recognized as safe) additive for food and feed applications. In general, dextran is used as gelling, viscosifying, texturing, and emulsifying agent in various food products (Leemhuis et al. 2013). Commercial applications of dextran from LAB are generally found in food and pharmaceutical industry; however, dextran also has several potential applications in photo film manufacturing, fine chemical, cosmetic, paper, petroleum, and textile industries (Naessens et al. 2005; Leemhuis et al. 2013). Due to the heterogeneity of dextran produced by various LAB , their application may depend on well-defined chemical and physicochemical properties. The properties of dextran that are applied in food industry are shown in Fig. 5.

Long-chain, high molecular weight polysaccharides that dissolve or disperse in water to give improved rheological (gelling, thickening) or physicochemical (emulsion stabilization, particle suspension, etc.) properties are important for food product formulation. A current consumer trend towards healthy and additive-free food has made the dextran an attractive food ingredient (Table 2). The microorganisms such as Leuconostoc mesenteroides, Saccharomyces cerevisiae, Lactobacillus plantarum, and Lactobacillus sanfrancisco are used for the production of dextran for its application in food processing without any restriction.

7.1 Bakery

The incorporation of dextran in bread for the improvement of rheological properties and quality is gaining interest (Galle et al. 2012; Wolter et al. 2014). The increasing knowledge of sourdough fermentation generates new opportunities for its use in the bakery field. In situ dextran production from Weissella sp. and Leuconostoc mesenteroides improved the freshness, mouthfeel, texture, loaf volume, softness, and shelf life of sourdough wheat bread (Katina et al. 2009; Galle et al. 2012). It came forth that dextran should have a high molecular weight and few branched linkages for the application in sourdough (Lacaze et al. 2007). The European Commission has approved the use of dextran in baked goods, up to the levels of 5 %. The addition of 2 % native dextran increases the water absorption of flour dough by about 12 %. However, in situ formation of dextran in sourdough was reported to be more effective than external addition (Brandt et al. 2003). High molecular weight dextrans of (1–2) × 106 Da have been approved by the European Union as food ingredients in bakery products (Naessens et al. 2005). The required molecular mass has been reported to be from 2 × 106 to about 4 × 106 Da (Katina et al. 2009).

Celiac disease is an autoimmune, nutrient-induced disorder, triggered in genetically susceptible individuals by ingesting gluten from wheat, rye, barley, and other closely related cereal grains (Goggins and Kelleher 1994). It was reported that celiac disease is a major health problem affecting around 1 % of population in the western world (Mustalahti et al. 2010). Currently, the only available treatment is the complete avoidance of gluten-containing cereals (Arendt et al. 2011). Gluten is an important protein-building structure which contributes to appearance and crumb structure in many bakery products. The replacement of gluten in bread presents a significant technological challenge due to the low nutritional quality, poor sensory characteristics such as dry crumb, poor mouth feel, and off flavors of gluten-free products (Galle et al. 2012; Hager and Arendt 2013). Hydrocolloids are currently used to substitute gluten and to obtain gluten-free bread with acceptable sensory properties (Galle et al. 2010; Hager and Arendt 2013). Incorporation of sourdough to a gluten-free formula gained interest recently in bread making. Dextran from Weissella and Leuconostoc species improves dough rheology and bread texture and can be used to replace nonbacterial hydrocolloids such as guar gum and hydroxypropyl methylcellulose for the generation of gluten-free soft bread with good texture and shelf life (Tieking and Ganzle 2005; Galle et al. 2012). Hence, dextran holds potential application in baking industry for the generation of gluten-free food products for patients suffering from celiac disease (Schwab et al. 2008; Galle et al. 2010; Rao and Goyal 2013).

7.2 Confectionery

Dextran is used for maintaining flavor, viscosity, moisture, inhibition of sugar crystallization, and as gelling agent in gum and jelly candies in confectioneries (Maina et al. 2011). It is also used in soft drinks, flavor extract, milk beverages, and icing.

7.3 Ice Cream

Dextran is also used as a cryoprotectant in ice cream (Naessens et al. 2005). Dextran is bland, odorless, tasteless, and nontoxic and is considered to have many advantages over other ice cream stabilizers. Ice cream mixes containing 2–4 % dextran conferred beneficial properties on viscosity (Bhavani and Nisha 2010).

7.4 Fermented Dairy Products

The texture of yogurt and yogurt-like products made from milk by fermentation with LAB can be modified by in situ production of EPS (Cerning 1995; Tamime and Robinson 1999). EPS produced by LAB, particularly dextran, positively affected the rheological properties of acidified milk gels with enhanced viscosity, creaminess, and reduced syneresis because of its water-binding ability (Mende et al. 2013) and hence can replace the commercially used texturizers, viz., xanthan, carrageenan, pectin, guar gum, and β-glucan.

7.5 Frozen Foods

The favorable properties of dextran for stabilizing vacuum, air-dried, and freeze-dried or frozen foods enable the use of dextran in fish products, meat, vegetables, and cheese. A film of dextran could protect food from oxidation and other chemical changes and also help to preserve texture and flavor. The increasing demand for fast food in frozen or dried state creates an opportunity for the use of dextran as a preservative, as well as a texture, flavor, and smell enhancer (Bhavani and Nisha 2010).

7.6 Reduced-Fat Cheese

The fat reduction in cheese results in many textural and functional defects. The high casein content in reduced-fat cheese imparts a firm and rubbery body and texture. Dextran is a good candidate for making reduced-fat cheese for several reasons. Dextran has the ability to bind water and increase the moisture in the non-fat mass (Awad et al. 2005).

7.7 Prebiotics

In recent years, there is a considerable interest in the use of prebiotics as functional foods in order to modulate the composition of the colonic microbiota to provide health benefits to the host (Saad et al. 2013). Foods containing prebiotic have also been associated with the protection against risk of several diseases, viz., bowel cancer, inflammatory bowel disease, diarrhea, coronary heart disease, obesity, osteoporosis, cholesterolemia, and type 2 diabetes. The α-(1 → 6) linkages are known to be resistant to hydrolysis by human intestinal enzymes, which results in the slow digestion of dextran in human. Moreover, α-(1 → 2) linkages are also highly resistant to the attack of digestive enzymes (Remaud-Simeon et al. 2000). Dextran and dextran-derived oligosaccharides have also been reported to increase the fraction of Bifidobacterium species in an in vitro model of the fermentation process in the human colon exhibiting prebiotic activity (Olano-Martin et al. 2000). A low molecular weight dextran containing α-(1 → 2)-branched linkages was also reported to act as prebiotic with selective effect on the gut microbiota (Sarbini et al. 2013). This dextran induced the growth of beneficial bacteria such as Bifidobacterium sp. and Lactobacillus sp. Recently, dextrans from Weissella cibaria JAG8 (Rao et al. 2014) and Lactobacillus plantarum DM5 (Das et al. 2014) showed promising prebiotic potential with very low gut digestibility and selective stimulation of probiotics.

7.8 Protein: Dextran Conjugates

Proteins are widely used in the food products such as beverages, yogurt, mayonnaise, and ice creams due to their functional properties, viz., emulsifying, foaming, gelling, and solubility (Oliver et al. 2006; Zhang et al. 2012). The functional properties of proteins can be improved by the conjugation of proteins and polysaccharides through Maillard reaction (Spotti et al. 2014). The Maillard reaction or nonenzymatic browning refers to any chemical reaction involving the interaction between amines and carbonyl compounds. Maillard reaction adds to the aroma, taste, and color of coffee and cocoa beans, bread, cakes, cereals, and meat (Martins et al. 2001). Dextran-conjugated proteins have displayed significant improvement in physical and chemical properties of proteins, such as thermal stability, emulsification, and antioxidant properties (Zhu et al. 2010). The improvement of functional properties of different proteins, such as ovalbumin, lysozyme (Chen et al. 2014), peanut protein (Liu et al. 2012), soy protein (Zhuo et al. 2013), and whey protein (Spotti et al. 2014), after conjugation with dextrans has been studied.

8 Side Effects of Dextran

Dextrans have acquired the GRAS status from the US FDA, which was renewed in 2013. However, several side effects are also associated with dextran polymer. Dextran is involved in the process of dental caries. Pathogenic bacteria of the genus Streptococcus produce insoluble dextran, which favors the adhesion of bacteria on the teeth and causes the formation of dental plaque. The orally ingested dextran rapidly converts into glucose, and therefore, it is harmful to the diabetic patients. The swelling or osmotic effect of dextran is also associated with acute renal failure in patients. It has also been reported that the high molecular weight dextran induces anti-dextran antibodies, leading to anaphylactoid reactions in some patients.

The production of dextran has been recognized as a nuisance to sugar industry for decades. Dextran formation creates processing problems in raw sugar factory operations and leads to a decline in sugar recovery, translating into economic loss. Dextran also creates processing difficulties by increasing juice viscosity, poor clarification, and crystal elongation. In addition, dextran has a significant impact on the resulting market on the final processed product. In some cases, dextran formation is also responsible for food spoilage in rum industry and cured meat products.

9 Conclusions

The sources, preparation, characterization, and food applications of dextran have been described. The biocompatibility, high water solubility, and water-holding capacity make dextran an important food ingredient. Therefore, the hunt for new, novel sources of dextran seems to be an interesting quest for food applications. Moreover, the production of dextran using cheaper substrates such as food wastes and agricultural by-products is prerequisite for its economical recovery at industrial level. Dextran conjugation has aided the design of new tailor-made polymers with different molecular weights, shapes, structures, and functional activities. It will be useful from an application viewpoint, if the functional properties of dextran, viz., rheology, molecular weight distribution, degree, and length of branching, and the fine structure of the dextran-conjugated products would be explored in greater details. The identification and characterization of novel dextransucrases with random mutagenesis followed by high-throughput screening will provide the best methods to obtain novel types of dextrans.